Vessel wall injury and artificial surfaces trigger sudden adhesion and aggregation of blood platelets, followed by the activation of the plasma coagulation system and the formation of fibrin-containing thrombi, which occlude the site of injury. These mechanisms are crucial to limit post-traumatic blood loss but may also occlude diseased vessels leading to ischemia and infarction of vital organs, or occlusion of CPB membranes.
In the classical waterfall model, blood coagulation proceeds by a series of reactions involving the activation of zymogens by limited proteolysis culminating in generation of thrombin, which converts plasma fibrinogen to fibrin and activates platelets. In turn, collagen- or fibrin-adherent platelets facilitate thrombin generation by several orders of magnitude via exposing procoagulant phospholipids (mainly phosphatidyl serine) on their outer surface, which propagates assembly and activation of coagulation protease complexes and by direct interaction between platelet receptors and coagulation factors.
Two converging pathways for coagulation exist that are triggered by either extrinsic (vessel wall) or intrinsic (blood-borne) components of the vascular system. The “extrinsic” pathway is initiated by the complex of the plasma factor VII (FVII) with the integral membrane protein tissue factor (TF), an essential coagulation cofactor that is absent on the luminal surface but strongly expressed in subendothelial layers of the vessel and which is accessible or liberated via tissue injury. TF expressed in circulating microvesicles might also contribute to thrombus propagation by sustaining thrombin generation on the surface of activated platelets.
The “intrinsic” or “contact activation pathway” is initiated when factor XII (FXII, Hageman factor) comes into contact with negatively charged surfaces in a reaction involving high molecular weight kininogen and plasma kallikrein. FXII can be activated by macromolecular constituents of the subendothelial matrix such as glycosaminoglycans and collagens, sulfatides, nucleotides and other soluble polyanions or non-physiological material such as glass or polymers. One of the most potent contact activators is kaolin and this reaction serves as the mechanistic basis for the major clinical clotting test, the activated partial thromboplastin time (aPTT), which measures the coagulation function of the “intrinsic” pathway. In reactions propagated by platelets, activated FXII (FXIIa) then activates FXI to FXIa and subsequently FXIa activates FIX to FIXa. The complex of FVIIIa, which FVIIIa has been previously activated by traces of FXa and/or thrombin, and FIXa (the tenase complex) subsequently activates FX to FXa. Despite its high potency to induce blood clotting in vitro, the (patho-) physiological significance of the FXII-triggered intrinsic coagulation pathway is questioned by the fact that hereditary deficiencies of FXII as well as of high molecular weight kininogen and plasma kallikrein are not associated with severe bleeding complications. Together with the observation that humans and mice lacking extrinsic pathway constituents such as TF and FVII suffer from severe bleeding this has led to the current hypothesis that for the cessation of bleeding in vivo exclusively the extrinsic cascade may be required (Mackman, N. 2004. Role of tissue factor in hemostasis, thrombosis, and vascular development. Arterioscler. Thromb. Vasc. Biol. 24, 101 5-1 022).
In pathological conditions, the coagulation cascade may be activated inappropriately which then results in the formation of hemostatically acting plugs inside the blood vessels. Thereby, vessels can be occluded and the blood supply to distal organs is limited. Furthermore, formed thrombi can detach and embolize into other parts of the body, there leading to ischemic occlusion. This process is known as thromboembolism and is associated with high mortality.
In WO20061066878, the use of antibodies against FXII/FXIIa or the use of inhibitors of FXII/FXIIa is proposed to prevent the formation and/or stabilization of thrombi. As potential inhibitors antithrombin (AT III), angiotensin converting enzyme inhibitor, C1 inhibitor, aprotinin, alpha-I protease inhibitor, antipain ([(S)-I-Carboxy-2-Phenylethyl]-Carbamoyl-L-Arg-L-Val-Arginal), Z-Pro-Proaldehyde-dimethyl acetate, DX88 (Dyax Inc., 300 Technology Square, Cambridge, Mass. 02139, USA; cited in: Williams A and Baird LG.2003. DX-88 and HAE: a developmental perspective. Transfus Apheresis Sci. 29:255-258), leupeptin, inhibitors of prolyl oligopeptidase such as Fmoc-Ala-Pyr-CN, corn-trypsin inhibitor, mutants of the bovine pancreatic trypsin inhibitor, ecotin, yellowfin sole anticoagulant protein, Cucurbita maxima trypsin inhibitor-v including Curcurbita maxima isoinhibitors and Hamadarin (as disclosed by Isawa H et al. 2002. A mosquito salivary protein inhibits activation of the plasma contact system by binding to factor XI and high molecular weight kininogen. J. Biol. Chem. 277:27651-27658) have been proposed.
Recently, Infestin-4 was reported to be a novel inhibitor of FXIIa. Infestins are a class of serine protease inhibitors derived from the midgut of the hematophagous insect, Triatoma infestans, a major vector for the parasite Trypanosoma cruzi, known to cause Chagas' disease (Campos ITN et al. 32 Insect Biochem. Mol. Bio. 991-997, 2002; Campos ITN et al. 577 FEBS Lett. 512-516, 2004). This insect uses these inhibitors to prevent coagulation of ingested blood. The Infestin gene encodes 4 domains that result in proteins that can inhibit different factors in the coagulation pathway. In particular, domain 4 encodes a protein (Infestin-4) that is a strong inhibitor of FXIIa. Infestin-4 has been administered in mice without bleeding complications (WO 2008/098720).
Because artificial surfaces can trigger the contact activation pathway there is a considerable medical risk involved in medical procedures which involve contacting blood with such artificial surfaces. Therefore the use of prosthetic devices or hemodialysers, which come into contact with blood, is severely limited because of activation of the intrinsic coagulation cascade. Suitable coating of the prosthetic surface may avoid said problem in some cases but may compromise its function in others. Examples of such prosthetic devices are heart valves, vascular stents and in-dwelling catheters.
In cases where such devices are used, anticoagulants, such as heparin, need to be administered to prevent fibrin formation on the surface.
However, some patients are intolerant of heparin, which can cause heparin-induced thrombocytopenia (HIT) resulting in platelet aggregation and life-threatening thrombosis. Furthermore, an inherent disadvantage of all anticoagulants used in clinics is an increased risk of serious bleeding events. Therefore, a strong need for new types of anticoagulants exist, which are not associated with such complications and that can be used in affected patients or as superior therapy concept preventing thrombosis without increased bleeding risks (Renne T et al. 2005. Defective thrombus formation in mice lacking factor XII. J. Exp. Med. 202:271-281).
A medical procedure involving a massive contact activation is cardiopulmonary bypass (CPB). Currently CPB devices in cardiac surgery are used for two reasons: a) artificial maintenance of the blood circulation during cardioplegia of patients undergoing heart surgery (pump function) and b) artificial oxygenation of the blood during cardioplegia of patients undergoing heart surgery (oxygenator function) via semipermeable membrane oxygenation.
Due to the artificial blood flow maintenance via the pump function the patient's blood is routed over a semipermeable membrane (˜3 m2, artificial surface) that allows oxygen passing through the membrane and binding to erythrocytes while the blood itself keeps in the closed artificial circulation system. This artificial oxygenation is vital to the patient and requires an anticoagulation strategy during CPB (H. P. Wendl at al 1996; Immunpharmacology; C. Sperling et al.; Biomaterials 30 (2009)). The currently used CPB devices have up to 3 m2 artificial surface that leads to a massive contact activation of the coagulation system, the inflammation system as well as activation of the complement system. In order to minimize these effects on the mentioned cascade systems, polymeric biomaterials of different kind are used (e.g. 2-methylacryloxyethylphosphorylcholin MCP) (Yu J et al.; Int J Artif Organs 1994; 17: 499-504; C. Sperling et al. Biomaterial 30 (2009) 4447-4456). Despite the fact that novel surface materials like polar phosphorylcholin which are less thrombogenic are used, it still remains necessary to anticoagulate patients undergoing CPB via heparin/bivalirudin since a platelet-mediated reaction would immediately lead to an occlusion of the CPB oxygenator with fatal outcome for the patient.
Currently there are two products licensed for anticoagulation during CPB:
a) Heparin:
Heparin is administered in body weight adjusted manner (300-400 IU/kg body weight (BW)) to the patient shortly before connecting the CPB devices in order to prevent the patient's blood from clotting. The CPB itself is also loaded with heparin before it is connected to the patient. This is the only anticoagulation strategy that allows CPB operations with an artificial oxygenation during the whole procedure without immediate fatal outcome for the patient. During this procedure the clotting capacity is monitored via activated clotting time (ACT) during the surgery (normal value 100-120 sec.; 300-500 sec. during operation). By measuring the ACT the physician can guide dosing of heparin in a semi-quantitative manner. Heparin binds AT (ATIII) and builds a fast inhibition complex that inactivates the coagulation system. Low molecular weight heparins (LMWH) mainly inhibit the prothrombinase-complex (factor X, factor Va, Ca2+, phospholipids) while unfractionated heparins (UFH) also inhibit factor II and therefore react faster than the LMWH. Furthermore, the factors IX, XI, XII and kallikrein are inactivated via heparins.
After the CPB the effect of heparin needs to be antagonized via protamine (1 mg/100 IU heparin) since a potential bleeding event might have a fatal outcome for the patient.
The major limitations of this standard of care are:
i) The amount of heparin in the patient's blood does not correlate with ACT (Gruenwald et al. 2010; the Journal of Extra Corporeal Technology) therefore it remains a risk for the patient to be either in a hypo- or hypercoagulopathic status.
ii) Heparin can induce thrombocypenia (HIT 1, caused by heparin via direct activation of thrombocytes, or HIT 2 caused by heparin and platelet factor 4 accumulations and consecutive development of antibodies against the complexes).
iii) The time window between heparin antagonization after CPB and the beginning of the anticoagulant therapy of patients on an intensive care unit (ICU) remains a risk factor for either bleeding, or thrombotic events.
iv) Protamine as antidote for the effect of heparin itself increases the risk for thrombotic events, severe allergic reactions and fatal drops of blood pressure. Further on a second administration of protamine in case of under dosing might have a fatal outcome for the patient.b) Bivalirudin:
Bivalirudin (derived from hirudin) is registered for the use in patients with known HIT as an alternative to anticoagulation via heparin. The limitations of this alternative therapy are:
i) No clinical registered antidote in case of bleeding is available at the moment.
ii) Higher consumption of blood products during CPB with resulting postoperative risks (C. Dyke et al.; surgery for acquired cardiovascular disease 2005)
In the present application it was surprisingly found that the use of inhibitors of FXII/FXIIa prevents clotting, while the risk for bleeding during and after CPB procedures is not enhanced in medical procedures which comprise contacting the blood of a patient with artificial surfaces, which in certain embodiments are outside of the body. Therefore only a reduced amount of other anticoagulants in addition to the FXII/FXIIa inhibitor needs to be administered. In one embodiment, the addition of other anticoagulants in addition to the FXII/FXIIa inhibitor can be completely avoided. This leads to safer medical procedures, as the increased bleeding risk which is inherent in the current therapy as well as complications by reversing the anticoagulation are avoided.